Intensive therapy for type 1 diabetes results in greater weight gain than conventional therapy. Many factors may predispose to this greater weight gain, including improved glycemic control, genetic susceptibility to obesity, and hypoglycemia. To study this, relationships among family history of type 2 diabetes, frequency of severe hypoglycemia, beta-cell autoantibodies, and weight gain were examined in 1,168 subjects aged > or =18 years at baseline randomized to intensive and conventional therapy groups in the Diabetes Control and Complications Trial. With intensive therapy, subjects with a family history of type 2 diabetes had greater central weight gain and dyslipidemia characterized by higher triglyceride levels and greater cholesterol in VLDLs and intermediate-density lipoproteins compared with subjects with no family history. Neither the frequency of severe hypoglycemia nor positivity to GAD65 and insulinoma-associated protein 2 antibodies was associated with increased weight gain with either intensive or conventional therapy. These data support the hypothesis that increased weight gain with intensive therapy might be explained, in part, by genetic traits.

To assess the role of insulin receptor (IR) substrate (IRS)-2 in insulin action and resistance in the liver, immortalized neonatal hepatocyte cell lines have been generated from IRS-2(-/-), IRS-2(+/-), and wild-type mice. These cells maintained the expression of the differentiated liver markers albumin and carbamoyl phosphate synthetase, as well as bear a high number of IRs. The lack of IRS-2 did not result in enhanced IRS-1 tyrosine phosphorylation or IRS-1-associated phosphatidylinositol (PI) 3-kinase activity on insulin stimulation. Total insulin-induced PI 3-kinase activity was decreased by 50% in IRS-2(-/-) hepatocytes, but the translocation of PI-3,4,5-trisphosphate to the plasma membrane in these cells was almost completely abolished. Downstream PI 3-kinase, activation of Akt, glycogen synthase kinase (GSK)-3 (alpha and beta isoforms), Foxo1, and atypical protein kinase C were blunted in insulin-stimulated IRS-2(-/-) cells. Reconstitution of IRS-2(-/-) hepatocytes with adenoviral IRS-2 restored activation of these pathways, demonstrating that IRS-2 is essential for functional insulin signaling in hepatocytes. Insulin induced a marked glycogen synthase activity in wild-type and heterozygous primary hepatocytes; interestingly, this response was absent in IRS-2(-/-) cells but was rescued by infection with adenoviral IRS-2. Regarding gluconeogenesis, the induction of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase by dibutyryl cAMP and dexamethasone was observed in primary hepatocytes of all genotypes. However, insulin was not able to suppress gluconeogenic gene expression in primary hepatocytes lacking IRS-2, but when IRS-2 signaling was reconstituted, these cells recovered this response to insulin. Suppression of gluconeogenic gene expression in IRS-2-deficient primary hepatocytes was also restored by infection with dominant negative Delta 256Foxo1.

HIV protease inhibitors (PIs) acutely and reversibly inhibit the insulin-responsive glucose transporter Glut 4, leading to peripheral insulin resistance and impaired glucose tolerance. Minimal modeling analysis of glucose tolerance tests on PI-treated patients has revealed an impaired insulin secretory response, suggesting additional pancreatic beta-cell dysfunction. To determine whether beta-cell function is acutely affected by PIs, we assayed glucose-stimulated insulin secretion in rodent islets and the insulinoma cell line MIN6. Insulin release from MIN6 cells and rodent islets was significantly inhibited by the PI indinavir with IC(50) values of 1.1 and 2.1 micro mol/l, respectively. The uptake of 2-deoxyglucose in MIN6 cells was similarly inhibited (IC(50) of 2.0 micro mol/l), whereas glucokinase activity was unaffected at drug levels as high as 1 mmol/l. Glucose utilization was also impaired at comparable drug levels. Insulin secretogogues acting downstream of glucose transport mostly reversed the indinavir-mediated inhibition of insulin release in MIN6 cells. Intravenous infusion of indinavir during hyperglycemic clamps on rats significantly suppressed the first-phase insulin response. These data suggest that therapeutic levels of PIs are sufficient to impair glucose sensing by beta-cells. Thus, together with peripheral insulin resistance, beta-cell dysfunction likely contributes to altered glucose homeostasis associated with highly active antiretroviral therapy.

The pyruvate dehydrogenase complex (PDC) is inactivated in many tissues during starvation and diabetes to conserve three-carbon compounds for gluconeogenesis. This is achieved by an increase in the extent of PDC phosphorylation caused in part by increased pyruvate dehydrogenase kinase (PDK) activity due to increased PDK expression. This study examined whether altered pyruvate dehydrogenase phosphatase (PDP) expression also contributes to changes in the phosphorylation state of PDC during starvation and diabetes. Of the two PDP isoforms expressed in mammalian tissues, the Ca(2+)-sensitive isoform (PDP1) is highly expressed in rat heart, brain, and testis and is detectable but less abundant in rat muscle, lung, kidney, liver, and spleen. The Ca(2+)-insensitive isoform (PDP2) is abundant in rat kidney, liver, heart, and brain and is detectable in spleen and lung. Starvation and streptozotocin-induced diabetes cause decreases in PDP2 mRNA abundance, PDP2 protein amount, and PDP activity in rat heart and kidney. Refeeding and insulin treatment effectively reversed these effects of starvation and diabetes, respectively. These findings indicate that opposite changes in expression of specific PDK and PDP isoenzymes contribute to hyperphosphorylation and therefore inactivation of the PDC in heart and kidney during starvation and diabetes.

Thiazolidinediones exert electrophysiologic effects in noncardiac cells in vitro, but to date there have been no reports of effects on cardiac rhythm. We previously demonstrated that chronic pretreatment with a thiazolidinedione peroxisome proliferator-activated receptor (PPAR)-gamma activator, troglitazone, improves recovery of left ventricular (LV) function and substrate metabolism after ischemia and reperfusion, without causing arrhythmias. In this study, we determined whether similar salutary effects are achieved with acute treatment with troglitazone. Anesthetized pigs underwent 90 min of regional LV ischemia and 90 min of reperfusion. Fifteen pigs were treated with troglitazone (10 mg/kg load, 5 mg. kg(-1). h(-1) infusion i.v.) beginning 1 h before ischemia. Seven pigs received corresponding vehicle. Plasma troglitazone concentration (mean 5 microg/ml) was similar to that achieved in clinical use of this agent. Before ischemia, acute troglitazone treatment had no effect on LV function, electrocardiogram, or substrate utilization. During ischemia or reperfusion, eight pigs in the troglitazone group died of ventricular fibrillation, compared with no pigs in the vehicle group (P < 0.05). Pigs that developed ventricular fibrillation had shorter QT intervals than survivors of either group. Among survivors, neither LV function nor substrate utilization differed between groups. Acute treatment with troglitazone increases susceptibility to ventricular fibrillation during myocardial ischemia and reperfusion. Whether thiazolidinediones have proarrhythmic potential in clinical use requires further investigation.

Catch-up growth is a risk factor for later obesity, type 2 diabetes, and cardiovascular diseases. We show here that after growth arrest by semistarvation, rats refed the same amount of a low-fat diet as controls show 1) lower energy expenditure due to diminished thermogenesis that favors accelerated fat deposition or catch-up fat and 2) normal glucose tolerance but higher plasma insulin after a glucose load at a time point when their body fat and plasma free fatty acids (FFAs) have not exceeded those of controls. Isocaloric refeeding on a high-fat diet resulted in even lower energy expenditure and thermogenesis and increased fat deposition and led to even higher plasma insulin and elevated plasma glucose after a glucose load. Stepwise regression analysis showed that plasma insulin and insulin-to-glucose ratio after the glucose load are predicted by variations in efficiency of energy use (i.e., in thermogenesis) rather than by the absolute amount of body fat or plasma FFAs. These studies suggest that suppression of thermogenesis per se may have a primary role in the development of hyperinsulinemia and insulin resistance during catch-up growth and underscore a role for suppressed thermogenesis directed specifically at catch-up fat in the link between catch-up growth and chronic metabolic diseases.

The metabolic syndrome is a cluster of metabolic and inflammatory abnormalities including obesity, insulin resistance, type 2 diabetes, hypertension, dyslipidemia, and atherosclerosis. The fatty acid binding proteins aP2 (fatty acid binding protein [FABP]-4) and mal1 (FABP5) are closely related and both are expressed in adipocytes. Previous studies in aP2-deficient mice have indicated a significant role for aP2 in obesity-related insulin resistance, type 2 diabetes, and atherosclerosis. However, the biological functions of mal1 are not known. Here, we report the generation of mice with targeted null mutations in the mal1 gene as well as transgenic mice overexpressing mal1 from the aP2 promoter/enhancer to address the role of this FABP in metabolic regulation in the presence or absence of obesity. To address the role of the second adipocyte FABP in metabolic regulation in the presence and deficiency of obesity, absence of mal1 resulted in increased systemic insulin sensitivity in two models of obesity and insulin resistance. Adipocytes isolated from mal1-deficient mice also exhibited enhanced insulin-stimulated glucose transport capacity. In contrast, mice expressing high levels of mal1 in adipose tissue display reduced systemic insulin sensitivity. Hence, our results demonstrate that mal1 modulates adipose tissue function and contributes to systemic glucose metabolism and constitutes a potential therapeutic target in insulin resistance.

Viral antibody-free BBDR and WF rats never develop spontaneous diabetes. BBDR rats, however, develop autoimmune diabetes after perturbation of the immune system, e.g., by viral infection. We previously identified a disease-susceptibility locus in the BBDR rat, iddm4, which is associated with the development of autoimmune diabetes after treatment with polyinosinic:polycytidylic acid and an antibody that depletes ART2(+) regulatory cells. We have now developed lines of congenic WF.iddm4 rats and report that in an intercross of N5 generation WF.iddm4 rats, approximately 70% of animals either homozygous or heterozygous for the BBDR origin allele of iddm4 became hyperglycemic after treatment to induce diabetes. Fewer than 20% of rats expressing the WF origin allele of iddm4 became diabetic. Testing the progeny of various recombinant N5 WF.iddm4 congenic rats for susceptibility to diabetes suggests that iddm4 is centered on a small segment of chromosome 4 bounded by the proximal marker D4Rat135 and the distal marker D4Got51, an interval of <2.8 cM. The allele at iddm4 has 79% sensitivity and 80% specificity in prediction of diabetes in rats that are segregating for this locus. These characteristics suggest that iddm4 is one of the most powerful non-major histocompatibility complex determinants of susceptibility to autoimmune diabetes described to date.

We investigated whether a polymorphism in codons 22 and 23 of the glucocorticoid (GC) receptor gene [GAGAGG(GluArg) --> GAAAAG(GluLys)] is associated with altered GC sensitivity, anthropometric parameters, cardiovascular risk factors, and sex steroid hormones. In a subgroup of 202 healthy elderly subjects of the Rotterdam Study, we identified 18 heterozygotes (8.9%) for the 22/23EK allele (ER22/23EK carriers). In the highest age group, the number of ER22/23EK carriers was higher (67-82 years, 12.9%) than in the youngest age group (53-67 years, 4.9%; P < 0.05). Two dexamethasone (DEX) suppression tests with 1 and 0.25 mg DEX were performed, and serum cortisol and insulin concentrations were compared between ER22/23EK carriers and noncarriers. After administration of 1 mg DEX, the ER22/23EK group had higher serum cortisol concentrations (54.8 +/- 18.3 vs. 26.4 +/- 1.4 nmol/l, P < 0.0001), as well as a smaller decrease in cortisol (467.0 +/- 31.7 vs. 484.5 +/- 10.3 nmol/l, P < 0.0001). ER22/23EK carriers had lower fasting insulin concentrations (P < 0.001), homeostasis model assessment- insulin resistance (IR) (index of IR, P < 0.05), and total (P < 0.02) and LDL cholesterol concentrations (P < 0.01). Our data suggest that carriers of the 22/23EK allele are relatively more resistant to the effects of GCs with respect to the sensitivity of the adrenal feedback mechanism than noncarriers, resulting in a better metabolic health profile.

Adipocyte factors play a major role in the induction of insulin resistance in skeletal muscle. To analyze this cross-talk, we established a system of co-culture of human fat and skeletal muscle cells. Cells of three muscle donors were kept in co-culture with cells of various fat cell donors, and insulin signaling was subsequently analyzed in myocytes. Insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was completely blocked, with unaltered expression of IRS-1. Troglitazone increased insulin action on IRS-1 phosphorylation, in both the absence and presence of co-culture. Insulin-regulated activation of Akt kinase in the myocytes was significantly reduced after co-culture, with troglitazone restoring insulin action. Addition of tumor necrosis factor (TNF)-alpha (2.5 nmol/l) to myocytes for 48 h reduced IRS-1 expression and inhibited IRS-1 and Akt phosphorylation comparable to the effect of co-culture. Lower doses of TNF-alpha were ineffective. After co-culture, TNF-alpha in the culture medium was below the detection limit of 0.3 pmol/l. A very low level of resistin was detected in the supernatant of myocytes, but not of adipocytes. In conclusion, the release of fat cell factors induces insulin resistance in human skeletal muscle cells; however, TNF-alpha and resistin appear not to be involved in this process.

Adiponectin, the most abundant adipose-specific protein, has been found to be negatively associated with degree of adiposity and positively associated with insulin sensitivity in Pima Indians and other populations. Moreover, adiponectin administration to rodents has been shown to increase insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and also increase whole-body insulin sensitivity. To further characterize the relationship between plasma adiponectin concentration and insulin sensitivity in humans, we examined 1) the cross-sectional association between plasma adiponectin concentration and skeletal muscle IR tyrosine phosphorylation and 2) the prospective effect of plasma adiponectin concentration at baseline on change in insulin sensitivity. Fasting plasma adiponectin concentration, body composition (hydrodensitometry or dual energy X-ray absorptiometry), insulin sensitivity (insulin-stimulated glucose disposal, hyperinsulinemic clamp), and glucose tolerance (75-g oral glucose tolerance test) were measured in 55 Pima Indians (47 men and 8 women, aged 31 +/- 8 years, body fat 29 +/- 8% [mean +/- SD]; 50 with normal glucose tolerance, 3 with impaired glucose tolerance, and 2 with diabetes). Group 1 (19 subjects) underwent skeletal muscle biopsies for the measurement of basal and insulin-stimulated tyrosine phosphorylation of the IR (stimulated by 100 nmol/l insulin). The fold increase after insulin stimulation was calculated as the ratio between maximal and basal phosphorylation. Group 2 (38 subjects) had follow-up measurements of insulin-stimulated glucose disposal. Cross-sectionally, plasma adiponectin concentration was positively associated with insulin-stimulated glucose disposal (r = 0.58, P < 0.0001) and negatively associated with percent body fat (r = -0.62, P < 0.0001) in the whole group. In group 1 plasma adiponectin was negatively associated with the basal (r = -0.65, P = 0.003) and positively associated with the fold increase in IR tyrosine phosphorylation (r = 0.69, P = 0.001) before and after the adjustment for percent body fat (r = -0.58, P = 0.01 and r = 0.54, P = 0.02, respectively). Longitudinally, after adjustment for age, sex, and percent body fat, low plasma adiponectin concentration at baseline was associated with a decrease in insulin sensitivity (P = 0.04). In conclusion, our cross-sectional data suggest a role of physiological concentration of fasting plasma adiponectin in the regulation of skeletal muscle IR tyrosine phosphorylation. Prospectively, low plasma adiponectin concentration at baseline precedes a decrease in insulin sensitivity. Our data indicate that adiponectin plays an important role in regulation of insulin sensitivity in humans.

Sterol regulatory element binding protein-1c (SREBP-1c) is a transcription factor that mediates insulin effects on hepatic gene expression. It is itself transcriptionally stimulated by insulin in hepatocytes. Here we show that SREBP-1c mRNA is expressed in adult rat skeletal muscles and that this expression is decreased by diabetes. The regulation of SREBP-1c expression was then assessed in cultures of adult muscle satellite cells. These cells form spontaneously contracting multinucleated myotubes within 7 days of culture. SREBP-1c mRNA is expressed in contracting myotubes. A 4-h treatment with 100 nmol/l insulin increases SREBP-1c expression and nuclear abundance by two- to threefold in myotubes. In cultured myotubes, insulin increases the expression of glycolytic and lipogenic enzyme genes and inhibits the 9-cis retinoic acid-induced UCP3 expression. These effects of insulin are mimicked by adenovirus-mediated expression of a transcriptionally active form of SREBP-1c. We conclude that in skeletal muscles, SREBP-1c expression is sensitive to insulin and can transduce the positive and negative actions of the hormone on specific genes and thus has a pivotal role in long-term muscle insulin sensitivity.

The potential role of the matrix metalloproteinase (MMP) system in the pathophysiology of the adipose tissue was investigated in a mouse model of nutritionally induced obesity. mRNA levels of 16 MMPs and 4 tissue inhibitors of MMPs (TIMPs) were measured by semiquantitative RT-PCR in adipose tissue isolated from mice maintained for 15 weeks on a standard or high-fat diet. In mice on standard diet, with the exception of MMP-8, all MMP and TIMP transcripts were detected in both gonadal and subcutaneous depots. In obese mice, the expression of MMP-3, -11, -12, -13, and -14 and TIMP-1 mRNAs was upregulated, whereas that of MMP-7, -9, -16, and -24 and TIMP-4 was downregulated. Most MMP and TIMP mRNAs were expressed at higher levels in stromal-vascular cells than in mature adipocytes. Analysis of adipose tissue by in situ fluorescent zymography revealed MMP-dependent proteolytic activities, demonstrating the presence of active MMPs in the intact tissue. In vitro conversion of adipogenic 3T3-F442A cells into mature adipocytes was associated with substantial modulations of MMP and TIMP expression. Moreover, this in vitro adipogenesis was reduced in the presence of a synthetic MMP inhibitor. Thus, the adipose tissue expresses a large array of MMPs and TIMPs, which modulate adipocyte differentiation.

Obesity is associated with insulin resistance, particularly when body fat has a central distribution. However, insulin resistance also frequently occurs in apparently lean individuals. It has been proposed that these lean insulin-resistant individuals have greater amounts of body fat than lean insulin-sensitive subjects. Alternatively, their body fat distribution may be different. Obesity is associated with elevated plasma leptin levels, but some studies have suggested that insulin sensitivity is an additional determinant of circulating leptin concentrations. To examine how body fat distribution contributes to insulin sensitivity and how these variables are related to leptin levels, we studied 174 individuals (73 men, 101 women), a priori classified as lean insulin-sensitive (LIS, n = 56), lean insulin-resistant (LIR, n = 61), and obese insulin-resistant (OIR, n = 57) based on their BMI and insulin sensitivity index (S(I)). Whereas the BMI of the two lean groups did not differ, the S(I) of the LIR subjects was less than half that of the LIS group. The subcutaneous and intra-abdominal fat areas, determined by computed tomography, were 45 and 70% greater in the LIR subjects (P < 0.001) and 2.5- and 3-fold greater in the OIR group, as compared with the LIS group. Fasting plasma leptin levels were moderately increased in LIR subjects (10.8 +/- 7.1 vs. 8.1 +/- 6.4 ng/ml in LIS subjects; P < 0.001) and doubled in OIR subjects (21.9 +/- 15.5 ng/ml; P < 0.001). Because of the confounding effect of body fat, we examined the relationships between adiposity, insulin sensitivity, and leptin concentrations by multiple regression analysis. Intra-abdominal fat was the best variable predicting insulin sensitivity in both genders and explained 54% of the variance in S(I). This inverse relationship was nonlinear (r = -0.688). On the other hand, in both genders, fasting leptin levels were strongly associated with subcutaneous fat area (r = 0.760) but not with intra-abdominal fat. In line with these analyses, when LIS and LIR subjects were matched for subcutaneous fat area, age, and gender, they had similar leptin levels, whereas their intra-abdominal fat and insulin sensitivity remained different. Thus, accumulation of intra-abdominal fat correlates with insulin resistance, whereas subcutaneous fat deposition correlates with circulating leptin levels. We conclude that the concurrent increase in these two metabolically distinct fat compartments is a major explanation for the association between insulin resistance and elevated circulating leptin concentrations in lean and obese subjects.

Type 1 diabetes has a substantial genetic component, with consistent evidence for a susceptibility locus in the HLA-DR/DQ region (chromosome 6p) and the insulin gene region (chromosome 11p). Genome scans have identified >18 other genomic regions that may harbor putative type 1 diabetes genes. However, evidence for most regions varies in different data sets. Given the genetic heterogeneity of type 1 diabetes, studies in homogeneous genetically isolated populations may be more successful in mapping susceptibility loci than in complex outbred populations. We describe a genome-wide search in a recently Dutch isolated population. We identified 43 patients that could be traced back to a common ancestor within 15 generations and performed a genome-wide scan using a combined linkage- and association-based approach. In addition to the HLA locus, evidence for type 1 diabetes loci was observed on chromosome 8q24 (marker D8S1128) and on chromosome 17q24 (marker D17S2059). Both the 8q and 17q localization are supported by allele-sharing at adjacent markers in affected individuals. Statistical evidence for a conserved ancestral haplotype was found for chromosome 8q24.

We examined the effect of three months of rosiglitazone treatment (4 mg b.i.d.) on whole-body insulin sensitivity and in vivo peripheral adipocyte insulin sensitivity as assessed by glycerol release in microdialysis from subcutaneous fat during a two-step (20 and 120 mU.m(-2).min(-1)) hyperinsulinemic-euglycemic clamp in nine type 2 diabetic subjects. In addition, the effects of rosiglitazone on liver and muscle triglyceride content were assessed by (1)H-nuclear magnetic resonance spectroscopy. Rosiglitazone treatment resulted in a 68% (P < 0.002) and a 20% (P < 0.016) improvement in insulin-stimulated glucose metabolism during the low- and high- dosage-insulin clamps, respectively, which was associated with approximately 40% reductions in plasma fatty acid concentration (P < 0.05) and hepatic triglyceride content (P < 0.05). These changes were associated with a 39% increase in extramyocellular lipid content (P < 0.05) and a 52% increase in the sensitivity of peripheral adipocytes to the inhibitory effects of insulin on lipolysis (P = 0.04). In conclusion, these results support the hypothesis that thiazolidinediones enhance insulin sensitivity in patients with type 2 diabetes by promoting increased insulin sensitivity in peripheral adipocytes, which results in lower plasma fatty acid concentrations and a redistribution of intracellular lipid from insulin responsive organs into peripheral adipocytes.

We studied the effects of hyperglycemia on beta-cell death and mass in syngeneically transplanted islets. Six groups of STZ-induced diabetic C57BL/6 mice were transplanted with 100 syngeneic islets, an insufficient beta-cell mass to restore normoglycemia. Groups 1, 2, and 3 remained hyperglycemic throughout the study. Groups 4, 5, and 6 were treated with insulin from day 7 before transplantation to day 10 after transplantation. After insulin discontinuation, group 6 mice achieved definitive normoglycemia. Grafts were harvested at 3 (groups 1 and 4), 10 (groups 2 and 5), and 30 (groups 3 and 6) days after transplantation. On day 3, the initially transplanted beta-cell mass (0.13 +/- 0.01 mg) was dramatically and similarly reduced in the hyperglycemic and insulin-treated groups (group 1: 0.048 +/- 0.002 mg; group 4: 0.046 +/- 0.007 mg; P < 0.001). Extensive islet necrosis (group 1: 30.7%; group 4: 26.8%) and increased beta-cell apoptosis (group 1: 0.30 +/- 0.05%; group 4: 0.42 +/- 0.07%) were found. On day 10, apoptosis remained increased in both hyperglycemic and insulin-treated mice (group 2: 0.44 +/- 0.09%; group 5: 0.48 +/- 0.08%) compared with normal pancreas (0.04 +/- 0.03%; P < 0.001). In contrast, on day 30, beta-cell apoptosis was increased in grafts exposed to sustained hyperglycemia (group 3: 0.37 +/- 0.03%) but not in normoglycemic mice (group 6: 0.12 +/- 0.02%); beta-cell mass was selectively reduced in islets exposed to hyperglycemia (group 3: 0.046 +/- 0.02 mg; group 6: 0.102 +/- 0.009 mg; P < 0.01). In summary, even in optimal conditions, approximately 60% of transplanted islet tissue was lost 3 days after syngeneic transplantation, and both apoptosis and necrosis contributed to beta-cell death. Increased apoptosis and reduced beta-cell mass were also found in islets exposed to chronic hyperglycemia, suggesting that sustained hyperglycemia increased apoptosis in transplanted beta-cells.

The metabolism of nitric oxide (NO) during cardiac surgery is unclear. We studied the effect of diabetes on NO metabolism during cardiac surgery in 40 subjects (20 with diabetes and 20 without diabetes). The patients were randomized to receive an infusion of physiological saline or nitroglycerin (GTN) at 1 microg. kg(-1). min(-1) starting 10 min before the initiation of cardiopulmonary bypass and then continuing for a period of 4 h. Blood and urine samples were collected at several time points for up to 8 h. NO metabolites were determined by the measurement of nitrate/nitrite (NOx, micromol/mmol creatinine) and cyclic guanosine monophosphate (cGMP, nmol/mmol creatinine) in plasma and urine. Plasma insulin levels were also determined at selected time points. Plasma NOx levels before surgery were significantly elevated in the group with diabetes compared with the group without diabetes (P < 0.001), and values were further increased during surgery in the former (P = 0.005) but not in the latter (P = 0.8). The greater plasma NOx values in patients with diabetes were matched by commensurate elevations in plasma cGMP levels (P = 0.01). Interestingly, infusion of GTN, an NO donor, significantly reduced plasma NOx (P < 0.001) and its urine elimination (P < 0.001) in patients with diabetes without reducing plasma cGMP levels (P = 0.89). Cardiac surgery increased plasma insulin in patients with and without diabetes; this increase was delayed by the infusion of GTN, but it was not related to the changes in NO production. In conclusion, NO production during cardiac surgery is increased in patients with diabetes, and this elevation can be blunted by the infusion of GTN in a rapid and reversible manner.

Ghrelin, a circulating growth hormone-releasing peptide derived from the stomach, stimulates food intake. The lowest systemically effective orexigenic dose of ghrelin was investigated and the resulting plasma ghrelin concentration was compared with that during fasting. The lowest dose of ghrelin that produced a significant stimulation of feeding after intraperitoneal injection was 1 nmol. The plasma ghrelin concentration after intraperitoneal injection of 1 nmol of ghrelin (2.83 +/- 0.13 pmol/ml at 60 min postinjection) was not significantly different from that occurring after a 24-h fast (2.79 +/- 0.32 pmol/ml). After microinjection into defined hypothalamic sites, ghrelin (30 pmol) stimulated food intake most markedly in the arcuate nucleus (Arc) (0-1 h food intake, 427 +/- 43% of control; P < 0.001 vs. control, P < 0.01 vs. all other nuclei), which is potentially accessible to the circulation. After chronic systemic or intracerebroventricular (ICV) administration of ghrelin for 7 days, cumulative food intake was increased (intraperitoneal ghrelin 13.6 +/- 3.4 g greater than saline-treated, P < 0.01; ICV ghrelin 19.6 +/- 5.5 g greater than saline-treated, P < 0.05). This was associated with excess weight gain (intraperitoneal ghrelin 21.7 +/- 1.4 g vs. saline 10.6 +/- 1.9 g, P < 0.001; ICV ghrelin 15.3 +/- 4.3 g vs. saline 2.2 +/- 3.8 g, P < 0.05) and adiposity. These data provide evidence that ghrelin is important in long-term control of food intake and body weight and that circulating ghrelin at fasting concentrations may stimulate food intake.

Postprandial release of the incretin glucagon-like peptide-1 (GLP-1) has been suggested to act as an endogenous satiety factor in humans. In rats, however, the evidence for this is equivocal probably because of very high endogenous activity of the GLP-1 degrading enzyme dipeptidyl peptidase-IV. In the present study, we show that intravenously administered GLP-1 (100 and 500 microg/kg) decreases food intake for 60 min in hungry rats. This effect is pharmacologically specific as it is inhibited by previous administration of 100 microg/kg exendin(9-39), and biologically inactive GLP-1(1-37) had no effect on food intake when administered alone (500 microg/kg). Acute intravenous administration of GLP-1 also caused dose-dependent inhibition of water intake, and this effect was equally well abolished by previous administration of exendin(9-39). A profound increase in diuresis was observed after intravenous administration of both 100 and 500 microg/kg GLP-1. Using a novel long-acting injectable GLP-1 derivative, NN2211, the acute and subchronic anorectic potentials of GLP-1 and derivatives were studied in both normal rats and rats made obese by neonatal monosodium glutamate treatment (MSG). We showed previously that MSG-treated animals are insensitive to the anorectic effects of centrally administered GLP-1(7-37). Both normal and MSG-lesioned rats were randomly assigned to groups to receive NN2211 or vehicle. A single bolus injection of NN2211 caused profound dose-dependent inhibition of overnight food and water intake and increased diuresis in both normal and MSG-treated rats. Subchronic multiple dosing of NN2211 (200 microg/kg) twice daily for 10 days to normal and MSG-treated rats caused profound inhibition of food intake. The marked decrease in food intake was accompanied by reduced body weight in both groups, which at its lowest stabilized at approximately 85% of initial body weight. Initial excursions in water intake and diuresis were transient as they were normalized within a few days of treatment. Lowered plasma levels of triglycerides and leptin were observed during NN2211 treatment in both normal and MSG-treated obese rats. In a subsequent study, a 7-day NN2211 treatment period of normal rats ended with measurement of energy expenditure (EE) and body composition determined by indirect calorimetry and dual energy X-ray absorptiometry, respectively. Compared with vehicle-treated rats, NN2211 and pair-fed rats decreased their total EE corresponding to the observed weight loss, such that EE per weight unit of lean body mass was unaffected. Despite its initial impact on body fluid balance, NN2211 had no debilitating effects on body water homeostasis as confirmed by analysis of body composition, plasma electrolytes, and hematocrit. This is in contrast to pair-fed animals, which displayed hemoconcentration and tendency toward increased percentage of fat mass. The present series of experiments show that GLP-1 is fully capable of inhibiting food intake in rats via a peripherally accessible site. The loss in body weight is accompanied by decreased levels of circulating leptin indicative of loss of body fat. The profound weight loss caused by NN2211 treatment was without detrimental effects on body water homeostasis. Thus, long-acting GLP-1 derivatives may prove efficient as weight-reducing therapeutic agents for overweight patients with type 2 diabetes.